Non-nucleotide containing enzymatic nucleic acid

ABSTRACT

Enzymatic nucleic acid molecule containing one or more non-nucleotide mimetics, and having activity to cleave an RNA or DNA molecule.

This application is a continuation of Usman et al., U.S. Ser. No.08/363,253, filed Dec. 23, 1994, now U.S. Pat. No. 5,891,683, which is acontinuation-in-part of Usman et al., U.S. Ser. No. 08/233,748 filedApr. 19, 1994 now abandoned, which is a continuation-in-part of Usman etal., U.S. Ser. No. 08/152,481, filed Nov. 12, 1993 now abandoned, whichis a continuation-in-part of Usman, U.S. Ser. No. 08/116,177, filed Sep.2, 1993 now abandoned, all entitled "Non-Nucleotide Containing EnzymaticNucleic Acid" and all hereby incorporated by reference herein (includingdrawings).

BACKGROUND OF THE INVENTION

This invention relates to chemically synthesizednon-nucleotide-containing enzymatic nucleic acid.

The following is a brief history of the discovery and activity ofenzymatic RNA molecules or ribozymes. This history is not meant to becomplete but is provided only for understanding of the invention thatfollows. This summary is not an admission that all of the work describedbelow is prior art to the claimed invention.

Prior to the 1970s it was thought that all genes were direct linearrepresentations of the proteins that they encoded. This simplistic viewimplied that all genes were like ticker tape messages, with each tripletof DNA "letters" representing one protein "word" in the translation.Protein synthesis occurred by first transcribing a gene from DNA intoRNA (letter for letter) and then translating the RNA into protein (threeletters at a time). In the mid 1970s it was discovered that some geneswere not exact, linear representations of the proteins that they encode.These genes were found to contain interruptions in the coding sequencewhich were removed from, or "spliced out" of, the RNA before it becametranslated into protein. These interruptions in the coding sequence weregiven the name of intervening sequences (or introns) and the process ofremoving them from the RNA was termed splicing. At least three differentmechanisms have been discovered for removing introns from RNA. Two ofthese splicing mechanisms involve the binding of multiple proteinfactors which then act to correctly cut and join the RNA. A thirdmechanism involves cutting and joining of the RNA by the intron itself,in what was the first discovery of catalytic RNA molecules.

Cech and colleagues were trying to understand how RNA splicing wasaccomplished in a single-celled pond organism called Tetrahymenathermophila. Cech proved that the intervening sequence RNA was acting asits own splicing factor to snip itself out of the surrounding RNA.Continuing studies in the early 1980's served to elucidate thecomplicated structure of the Tetrahymena intron and to decipher themechanism by which self-splicing occurs. Many research groups helped todemonstrate that the specific folding of the Tetrahymena intron iscritical for bringing together the parts of the RNA that will be cut andspliced. Even after splicing is complete, the released intron maintainsits catalytic structure. As a consequence, the released intron iscapable of carrying out additional cleavage and splicing reactions onitself (to form intron circles). By 1986, Cech was able to show that ashortened form of the Tetrahymena intron could carry out a variety ofcuffing and joining reactions on other pieces of RNA. The demonstrationproved that the Tetrahymena intron can act as a true enzyme: (i) eachintron molecule was able to cut many substrate molecules while theintron molecule remained unchanged, and (ii) reactions were specific forRNA molecules that contained a unique sequence (CUCU) which allowed theintron to recognize and bind the RNA. Zaug and Cech coined the term"ribozyme" to describe any ribonucleic acid molecule that hasenzyme-like properties.

Also in 1986, Cech showed that the RNA substrate sequence recognized bythe Tetrahymena ribozyme could be changed by altering a sequence withinthe ribozyme itself. This property has led to the development of anumber of site-specific ribozymes that have been individually designedto cleave at other RNA sequences.

The Tetrahymena intron is the most well-studied of what is nowrecognized as a large class of introns, Group I introns. The overallfolded structure, including several sequence elements, is conservedamong the Group I introns, as is the general mechanism of splicing. Likethe Tetrahymena intron, some members of this class are catalytic, i.e.,the intron itself is capable of the self-splicing reaction. Other GroupI introns require additional (protein) factors, presumably to help theintron fold into and/or maintain its active structure.

Ribonuclease P (RNaseP) is an enzyme comprised of both RNA and proteincomponents which are responsible for converting precursor tRNA moleculesinto their final form by trimming extra RNA off one of their ends.RNaseP activity has been found in all organisms tested. Sidney Altmanand his colleagues showed that the RNA component of RNaseP is essentialfor its processing activity; however, they also showed that the proteincomponent also was required for processing under their experimentalconditions. After Cech's discovery of self-splicing by the Tetrahymenaintron, the requirement for both protein and RNA components in RNasePwas reexamined. In 1983, Altman and Pace showed that the RNA was theenzymatic component of the RNaseP complex. This demonstrated that an RNAmolecule was capable of acting as a true enzyme, processing numeroustRNA molecules without itself undergoing any change.

The folded structure of RNaseP RNA has been determined, and while thesequence is not strictly conserved between RNAs from differentorganisms, this higher order structure is. It is thought that theprotein component of the RNaseP complex may serve to stabilize thefolded RNA in vivo.

Symons and colleagues identified two examples of a self-cleaving RNAthat differed from other forms of catalytic RNA already reported. Symonswas studying the propagation of the avocado sunblotch viroid (ASV), anRNA virus that infects avocado plants. Symons demonstrated that aslittle as 55 nucleotides of the ASV RNA was capable of folding in such away as to cut itself into two pieces. It is thought that in vivoself-cleavage of these RNAs is responsible for cutting the RNA intosingle genome-length pieces during viral propagation. Symons discoveredthat variations on the minimal catalytic sequence from ASV could befound in a number of other plant pathogenic RNAs as well. Comparison ofthese sequences' revealed a common structural design consisting of threestems and loops connected by a central loop containing many conserved(invariant from one RNA to the next) nucleotides. The predictedsecondary structure for this catalytic RNA reminded the researchers ofthe head of a hammer; thus it was named as such.

Uhlenbeck was successful in separating the catalytic region of theribozyme from that of the substrate. Thus, it became possible toassemble a hammerhead ribozyme from 2 (or 3) small synthetic RNAs. A19-nucleotide catalytic region and a 24-nucleotide substrate weresufficient to support specific cleavage. The catalytic domain ofnumerous hammerhead ribozymes have now been studied by both theUhlenbeck's and Symons' groups with regard to defining the nucleotidesrequired for specific assembly and catalytic activity, and determiningthe rates of cleavage under various conditions.

Haseloff and Gerlach showed it was possible to divide the domains of thehammerhead ribozyme in a different manner. By doing so, they placed mostof the required sequences in the strand that did not get cut (theribozyme) and only a required UH where H=C, A, or U in the strand thatdid get cut (the substrate). This resulted in a catalytic ribozyme thatcould be designed to cleave any UH RNA sequence embedded within a longer"substrate recognition" sequence. The specific cleavage of a long mRNA,in a predictable manner using several such hammerhead ribozymes, wasreported in 1988.

One plant pathogen RNA (from the negative strand of the tobacco ringspotvirus) undergoes self-cleavage but cannot be folded into the consensushammerhead structure described above. Bruening and colleagues haveindependently identified a 50-nucleotide catalytic domain for this RNA.In 1990, Hampel and Tritz succeeded in dividing the catalytic domaininto two parts that could act as substrate and ribozyme in amultiple-turnover, cutting reaction. As with the hammerhead ribozyme,the catalytic portion contains most of the sequences required forcatalytic activity, while only a short sequence (GUC in this case) isrequired in the target. Hampel and Tritz described the folded structureof this RNA as consisting of a single hairpin and coined the term"hairpin" ribozyme (Bruening and colleagues use the term "paperclip" forthis ribozyme motif). Continuing experiments suggest an increasingnumber of similarities between the hairpin and hammerhead ribozymes inrespect to both binding of target RNA and mechanism of cleavage.

Hepatitis Delta Virus (HDV) is a virus whose genome consists ofsingle-stranded RNA. A small region (about 80 nucleotides) in both thegenomic RNA, and in the complementary anti-genomic RNA, is sufficient tosupport self-cleavage. In 1991, Been and Perrotta proposed a secondarystructure for the HDV RNAs that is conserved between the genomic andanti-genomic RNAs and is necessary for catalytic activity. Separation ofthe HDV RNA into "ribozyme" and "substrate" portions has recently beenachieved by Been. Been has also succeeded in reducing the size of theHDV ribozyme to about 60 nucleotides.

Table I lists some of the characteristics of the ribozymes discussedabove.

Eckstein et al., International Publication No. WO 92/07065; Perrault etal., Nature 1990, 344:565; Pieken et al., Science 1991, 253:314; Usmanand Cedergren, Trends in Biochem. Sci. 1992, 17:334; and Rossi et al.,International Publication No. WO 91/03162, describe various chemicalmodifications that can be made to the sugar moieties of enzymaticnucleic acid molecules.

Usman, et al., WO 93/15187 in discussing modified structures inribozymes states:

It should be understood that the linkages between the building units ofthe polymeric chain may be linkages capable of bridging the unitstogether for either in vitro or in vivo. For example the linkage may bea phosphorous containing linkage, e.g., phosphodiester orphosphothioate, or may be a nitrogen containing linkage, e.g., amide. Itshould further be understood that the chimeric polymer may containnon-nucleotide spacer molecules along with its other nucleotide oranalogue units. Examples of spacer molecules which may be used aredescribed in Nielsen et al. Science, 254:1497-1500 (1991).

Jennings et al., WO 94/13688 while discussing hammerhead ribozymeslacking the usual stem II base-paired region state:

One or more ribonucleotides and/or deoxyribonucleotides of the group(X)_(m), [stem II] may be replaced, for example, with a linker selectedfrom optionally substituted polyphosphodiester (such aspoly(1-phospho-3-propanol)), optionally substituted alkyl, optionallysubstituted polyamide, optionally substituted glycol, and the like.Optional substituents are well known in the art, and include alkoxy(such as methoxy, ethoxy and propoxy), straight or branch chain loweralkyl such as C₁ -C₅ alkyl), amine, aminoalkyl (such as amino C₁ -C₅alkyl), halogen (such as F, Cl and Br) and the like. The nature ofoptional substituents is not of importance, as long as the resultantendonuclease is capable of substrate cleavage.

Additionally, suitable linkers may comprise polycyclic molecules, suchas those containing phenyl or cyclohexyl rings. The linker (L) may be apolyether such as polyphosphopropanediol, polyethyleneglycol, abifunctional polycyclic molecule such as a bifunctional pentalene,indene, naphthalene, azulene, heptalene, biphenylene, asymindacene,sym-indacene, acenaphthylene, fluorene, phenalene, phenanthrene,anthracene, fluoranthene, acephenathrylene, aceanthrylene, triphenylene,pyrene, chrysene, naphthacene, thianthrene, isobenzofuran, chromene,xanthene, phenoxathiin, indolizine, isoindole, 3-H-indole, indole,1-H-indazole, 4-H-quinolizine, isoquinoline, quinoline, phthalazine,naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine,4-αH-carbzole, carbazole, B-carboline, phenanthridine, acridine,perimidine, phenanthroline, phenazine, phenolthiazine, phenoxazine,which polycyclic compound may be substituted or modified, or acombination of the polyethers and the polycyclic molecules.

The polycyclic molecule may be substituted of polysubstituted with C₁-C₅ alkyl, alkenyl, hydroxyalkyl, halogen of haloalkyl group or withO--A or CH₂ --O--A wherein A is H or has the formula CONR'R" wherein R'and R" are the same or different and are hydrogen or a substituted orunsubstituted C₁ -C₆ alkyl, aryl, cycloalkyl, or heterocyclic group; orA has the formula --M--NR'R" wherein R' and R" are the same or differentand are hydrogen, or a C₁ -C₅ alkyl, alkenyl, hydroxyalkyl, or haloalkylgroup wherein the halo atom is fluorine, chlorine, bromine, or iodineatom; and --M-- is an organic moiety having 1 to 10 carbon atoms and isa branched or straight chain alkyl, aryl, or cycloalkyl group.

In one embodiment, the linker is tetraphosphopropanediol orpentaphosphopropanediol. In the case of polycyclic molecules there willbe preferably 18 or more atoms bridging the nucleic acids. Morepreferably their will be from 30 to 50 atoms bridging, see for Example5. In another embodiment the linker is a bifunctional carbazole orbifunctional carbazole linked to one or more polyphosphoropropanediol.

Such compounds may also comprise suitable functional groups to allowcoupling through reactive groups on nucleotides."

SUMMARY OF THE INVENTION

This invention concerns the use of non-nucleotide molecules as spacerelements at the base of double-stranded nucleic acid (e.g., RNA or DNA)stems (duplex stems) or more preferably, in the single-stranded regions,catalytic core, loops, or recognition arms of enzymatic nucleic acids.Duplex stems are ubiquitous structural elements in enzymatic RNAmolecules. To facilitate the synthesis of such stems, which are usuallyconnected via single-stranded nucleotide chains, a base or base-pairmimetic may be used to reduce the nucleotide requirement in thesynthesis of such molecules, and to confer nuclease resistance (sincethey are non-nucleic acid components). This also applies to both thecatalytic core and recognition arms of a ribozyme. In particular abasicnucleotides (i.e., moieties lacking a nucleotide base, but having thesugar and phosphate portions) can be used to provide stability within acore of a ribozyme, e.g., at U4 or N7 or a hammerhead structure shown inFIG. 1.

Thus, in a first aspect, the invention features an enzymatic nucleicacid molecule having one or more non-nucleotide moieties, and havingenzymatic activity to cleave an RNA or DNA molecule.

Examples of such non-nucleotide mimetics are shown in FIG. 6 and theirincorporation into hammerhead ribozymes is shown in FIG. 7. Thesenon-nucleotide linkers may be either polyether, polyamine, polyamide, orpolyhydrocarbon compounds. Specific examples include those described bySeela and Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic AcidsRes. 1987, 15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991,113:6324; Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Maet al., Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751;Durand et al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al.,Nucleosides & Nucleotides 1991, 10:287; Jaschke et al., TetrahedronLett. 1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold etal., International Publication No. WO 89/02439 entitled "Non-nucleotideLinking Reagents for Nucleotide Probes"; and Ferentz and Verdine, J. Am.Chem. Soc. 1991, 113:4000, all hereby incorporated by reference herein.

In preferred embodiments, the enzymatic nucleic acid includes one ormore stretches of RNA, which provide the enzymatic activity of themolecule, linked to the non-nucleotide moiety.

By the term "non-nucleotide" is meant any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound is abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenine, guanine, cytosine, uracilor thymine. It may have substitutions for a 2' or 3' H or OH asdescribed in the art. See Eckstein et al. and Usman et al., supra.

In preferred embodiments, the enzymatic nucleic acid includes one ormore stretches of RNA, which provide the enzymatic activity of themolecule, linked to the non-nucleotide moiety. The necessaryribonucleotide components are known in the art, see, e.g., Usman, supraand Usman et al., Nucl. Acid. Symp. Genes 31:163, 1994.

As the term is used in this application, non-nucleotide-containingenzymatic nucleic acid means a nucleic acid molecule that contains atleast one non-nucleotide component which replaces a portion of aribozyme, e.g., but not limited to, a double-stranded stem, asingle-stranded "catalytic core" sequence, a single-stranded loop or asingle-stranded recognition sequence. These molecules are able to cleave(preferably, repeatedly cleave) separate RNA or DNA molecules in anucleotide base sequence specific manner. Such molecules can also act tocleave intramolecularly if that is desired. Such enzymatic molecules canbe targeted to virtually any RNA transcript. Such molecules also includenucleic acid molecules having a 3' or 5' non-nucleotide, useful as acapping group to prevent exonuclease digestion.

Enzymatic molecules of this invention act by first binding to a targetRNA or DNA. Such binding occurs through the target binding portion ofthe enzyme which is held in close proximity to an enzymatic portion ofmolecule that acts to cleave the target RNA or DNA. Thus, the moleculefirst recognizes and then binds a target nucleic acid throughcomplementary base-pairing, and once bound to the correct site, actsenzymatically to cut the target. Strategic cleavage of such a targetwill destroy its ability to direct synthesis of an encoded protein.After an enzyme of this invention has bound and cleaved its target it isreleased from that target to search for another target, and canrepeatedly bind and cleave new targets.

The enzymatic nature of an enzyme of this invention is advantageous overother technologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the effective concentration of the enzyme necessary to effect atherapeutic treatment is lower than that of an antisenseoligonucleotide. This advantage reflects the ability of the enzyme toact enzymatically. Thus, a single enzyme molecule is able to cleave manymolecules of target RNA. In addition, the enzyme is a highly specificinhibitor, with the specificity of inhibition depending not only on thebase pairing mechanism of binding, but also on the mechanism by whichthe molecule inhibits the expression of the RNA to which it binds. Thatis, the inhibition is caused by cleavage of the target and sospecificity is defined as the ratio of the rate of cleavage of thetargeted nucleic acid over the rate of cleavage of non-targeted nucleicacid. This cleavage mechanism is dependent upon factors additional tothose involved in base pairing. Thus, it is thought that the specificityof action of an enzyme of this invention is greater than that ofantisense oligonucleotide binding the same target site.

By "complementarity" is meant a nucleic acid that can form hydrogenbond(s) with other RNA sequence by either traditional Watson-Crick orother non-traditional types (for example, Hoogsteen type) of base-pairedinteractions.

By the phrase enzyme is meant a catalytic non-nucleotide-containingnucleic acid molecule that has complementarity in a substrate-bindingregion to a specified nucleic acid target, and also has an enzymaticactivity that specifically cleaves RNA or DNA in that target. That is,the enzyme is able to intramolecularly or intermolecularly cleave RNA orDNA and thereby inactivate a target RNA or DNA molecule. Thiscomplementarity functions to allow sufficient hybridization of theenzymatic molecule to the target RNA or DNA to allow the cleavage tooccur. One hundred percent complementarity is preferred, butcomplementarity as low as 50-75% may also be useful in this invention.

In preferred embodiments of this invention, the enzymatic nucleic acidmolecule is formed in a hammerhead or hairpin motif, but may also beformed in the motif of a hepatitis delta virus, group I intron or RNasePRNA (in association with an RNA guide sequence) or Neurospora VS RNA.Examples of such hammerhead motifs are described by Rossi et al., 1992,Aids Research and Human Retroviruses 8, 183, of hairpin motifs by Hampelet al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, andHampel at al., 1990 Nucleic Acids Res. 18, 299, and an example of thehepatitis delta virus motif is described by Perrotta and Been, 1992Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al., 1983Cell 35, 849, Neurospora VS RNA ribozyme motif is described by Collins(Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993Biochemistry 32, 2795-2799) and of the Group I intron by Cech et al.,U.S. Pat. No. 4,987,071. These specific motifs are not limiting in theinvention and those skilled in the art will recognize that all that isimportant in an enzymatic nucleic acid molecule of this invention isthat it has a specific substrate binding site which is complementary toone or more of the target gene RNA regions, and that it have nucleotidesequences within or surrounding that substrate binding site which impartan RNA cleaving activity to the molecule.

The invention provides a method for producing a class of enzymaticcleaving agents which exhibit a high degree of specificity for thenucleic acid of a desired target. The enzyme molecule is preferablytargeted to a highly conserved sequence region of a target such thatspecific treatment of a disease or condition can be provided with asingle enzyme. Such enzyme molecules can be delivered exogenously tospecific cells as required. In the preferred hammerhead motif the smallsize (less than 60 nucleotides, preferably between 30-40 nucleotides inlength) of the molecule allows the cost of treatment to be reducedcompared to other ribozyme motifs.

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small enzyme motifs (e.g.,of the hammerhead structure) are used for exogenous delivery. The simplestructure of these molecules increases the ability of the enzyme toinvade targeted regions of mRNA structure. Unlike the situation when thehammerhead structure is included within longer transcripts, there are nonon-enzyme flanking sequences to interfere with correct folding of theenzyme structure or with complementary regions.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawings will first briefly be described.

Drawings:

FIG. 1 is a diagrammatic representation of a hammerhead ribozyme domainknown in the art. Stem II can be ≧2 base-pair long, or can even lackbase pairs and consist of a loop region.

FIG. 2a is a diagrammatic representation of the hammerhead ribozymedomain known in the art; FIG. 2b is a diagrammatic representation of thehammerhead ribozyme as divided by Uhlenbeck (1987, Nature, 327, 596)into a substrate and enzyme portion; FIG. 2c is a similar diagramshowing the hammerhead divided by Haseloff and Gerlach (1988, Nature,334, 585) into two portions; and FIG. 2d is a similar diagram showingthe hammerhead divided by Jeffries and Symons (1989, Nucleic. Acids.Res., 17, 1371) into two portions.

FIG. 3 is a diagrammatic representation of the general structure of ahairpin ribozyme. Helix 2 (H2) is provided with a least 4 base pairs(i.e., n is 1, 2, 3 or 4) and helix 5 can be optionally provided oflength 2 or more bases (preferably 3-20 bases, i.e., m is from 1-20 ormore). Helix 2 and helix 5 may be covalently linked by one or more bases(i.e., r is ≧1 base). Helix 1, 4 or 5 may also be extended by 2 or morebase pairs (e.g., 4-20 base pairs) to stabilize the ribozyme structure,and preferably is a protein binding site. In each instance, each N andN' independently is any normal or modified base and each dash representsa potential base-pairing interaction. These nucleotides may be modifiedat the sugar, base or phosphate. Complete base-pairing is not requiredin the helices, but is preferred. Helix 1 and 4 can be of any size(i.e., o and p is each independently from 0 to any number, e.g., 20) aslong as some base-pairing is maintained. Essential bases are shown asspecific bases in the structure, but those in the art will recognizethat one or more may be modified chemically (abasic, base, sugar and/orphosphate modifications) or replaced with another base withoutsignificant effect. Helix 4 can be formed from two separate molecules,i.e., without a connecting loop. The connecting loop when present may bea ribonucleotide with or without modifications to its base, sugar orphosphate. "q" is ≧2 bases. The connecting loop can also be replacedwith a non-nucleotide linker molecule. H , refers to bases A, U or C. Yrefers to pyrimidine bases. "₋₋₋₋ " refers to a chemical bond.

FIG. 4 is a representation of the general structure of the hepatitisdelta virus ribozyme domain known in the art (Perrota and Been, 1991supra).

FIG. 5A is a representation of the general structure of theself-cleaving Neurospora VS RNA domain. FIG. 5B is a line diagramrepresenting the "I" ribozyme motif. The figure shows the "Upper" andthe "Lower" base-paired regions linked by the "connecting" region. IV(left) and V (right) shows the left and the right handed regions withinthe "upper" region, respectively. II (left) and VI (right) shows theleft and the right handed regions within the "lower" region,respectively).

FIGS. 6A and 6B are diagrammatic representations of variousnon-nucleotide mimetics that may be incorporated into nucleic acidenzymes. Standard abbreviations are used in the Figure. In compound 1each X may independently be oxygen, nitrogen, sulfur or substitutedcarbons containing alkyl, alkene or equivalent chains of length 1-10carbon atoms. In compounds 6, 6a, 7, 8, 9 and 10 each Y mayindependently be a phosphodiester, ether or amide linkage to the rest ofthe nucleic acid enzyme. In compounds 4 and 5 each R may independentlybe H, OH, protected OH, O-alkyl, alkenyl or alkynyl or alkyl, alkenyl oralkynyl of 1-10 carbon atoms.

FIG. 7 is a diagrammatic representation of the preferred location forincorporation of various non-nucleotide mimetics into nucleic acidenzymes. Specifically, mimetics, 1-10, may replace the loop (denoted asin FIG. 7) that connects the two strands of Stem II. Stem II itself maybe from 1 to 10 base pairs. In examples 1 & 2 below compounds 1 and 2were incorporated into molecules having a stem II of 1 to 5 basepairs inlength. Compounds 1, 4 and 5 may also replace nucleotides in therecognition arms of stems I and III or in stem II itself.

FIG. 8 is a diagrammatic representation of the synthesis of a perylenebased non-nucleotide mimetic phosphoramidite 3.

FIGS. 9A and 9B are diagrammatic representations of the synthesis of anabasic deoxyribose or ribose non-nucleotide mimetic phosphoramidite.

FIGS. 10a and 10b are graphical representations of cleavage of substrateby various ribozymes at 8 nM, or 40 nM, respectively.

FIG. 11 is a diagrammatic representation of a hammerhead ribozymetargeted to site A (HHA). Arrow indicates the cleavage site. Stem II isshorter than usual for a hammerhead ribozyme.

FIG. 12 is a diagrammatic representation of HHA ribozyme containingabasic substitutions (HHA-a) at various positions. Ribozymes weresynthesized as described in the application. "X" shows the positions ofabasic substitutions. The abasic substitutions were either madeindividually or in certain combinations.

FIG. 13 shows the in vitro RNA cleavage activity of HHA and HHA-aribozymes. All RNA, refers to HHA ribozyme containing no abasicsubstitution. U4 Abasic, refers to HHA-a ribozyme with a single abasic(ribose) substitution at position 4. U7 Abasic, refers to HHA-a ribozymewith a single abasic (ribose) substitution at position 7.

FIG. 14 shows in vitro RNA cleavage activity of HHA and HHA-a ribozymes.Abasic Stem II Loop, refers to HHA-a ribozyme with four abasic (ribose)substitutions within the loop in stem II.

FIG. 15 shows in vitro RNA cleavage activity of HHA and HHA-a ribozymes.3'-lnverted Deoxyribose, refers to HHA-a ribozyme with an inverteddeoxyribose (abasic) substitution at its 3' termini.

FIG. 16 is a diagrammatic representation of a hammerhead ribozymetargeted to site B (HHB). Target B is involved in the proliferation ofmammalian smooth muscle cells. Arrow indicates the site of cleavage.Inactive version of HHB contains 2 base-substitutions (G5U and A15.1U)that renders the ribozyme catalytically inactive.

FIG. 17 is a diagrammatic representation of HHB ribozyme with abasicsubstitution (HHB-a) at position 4. X, shows the position of abasicsubstitution.

FIG. 18 shows ribozyme-mediated inhibition of rat aortic smooth musclecell (RASMC) proliferation. Both HHB and HHB-a ribozymes can inhibit theproliferation of RASMC in culture. Catalytically inactive HHB ribozymeshows inhibition which is significantly lower than active HHB and HHB-aribozymes.

NON-NUCLEOTIDE MIMETICS

Non-nucleotide mimetics useful in this invention are generally describedabove. Those in the art will recognize that these mimetics can beincorporated into an enzymatic molecule by standard techniques at anydesired location. Suitable choices can be made by standard experimentsto determine the best location, e.g., by synthesis of the molecule andtesting of its enzymatic activity. The optimum molecule will contain theknown ribonucleotides needed for enzymatic activity, and will havenon-nucleotides which change the structure of the molecule in the leastway possible. What is desired is that several nucleotides can besubstituted by one non-nucleotide to save synthetic steps in enzymaticmolecule synthesis and to provide enhanced stability of the moleculecompared to RNA or even DNA.

Synthesis of Ribozymes

In this invention, small enzymatic nucleic acid motifs (e.g., of thehammerhead or the hairpin structure) are used for exogenous delivery.The simple structure of these molecules increases the ability of theenzymatic nucleic acid to invade targeted regions of the mRNA structure.The ribozymes are chemically synthesized. The method of synthesis usedfollows the procedure for normal RNA synthesis as described in Usman etal., 1987 J. Am. Chem. Soc., 109, 7845 and in Scaringe et al., 1990Nucleic Acids Res., 18, 5433 and makes use of common nucleic acidprotecting and coupling groups, such as dimethoxytrityl at the 5'-end,and phosphoramidites at the 3'-end. The average stepwise coupling yieldswere >98%.

Ribozymes are purified by gel electrophoresis using general methods orare purified by high pressure liquid chromatography (HPLC; See Usman etal., Synthesis, deprotection, analysis and purification of RNA andribozymes, filed May, 18, 1994, U.S. Ser. No. 08/245,736 the totality ofwhich is hereby incorporated herein by reference) and are resuspended inwater.

Various modifications to ribozyme structure can be made to enhance theutility of ribozymes. Such modifications will enhance shelf-life,half-life in vitro, stability, and ease of introduction of suchribozymes to the target site, e.g., to enhance penetration of cellularmembranes, and confer the ability to recognize and bind to targetedcells.

Optimizing Ribozyme Activity

Ribozyme activity can be optimized as described by Stinchcomb et al.,"Method and Composition for Treatment of Restenosis and Cancer UsingRibozymes," filed May 18, 1994, U.S. Ser. No. 08/245,466. The detailswill not be repeated here, but include altering the length of theribozyme binding arms (stems I and III, see FIG. 2c), or chemicallysynthesizing ribozymes with modifications that prevent their degradationby serum ribonucleases (see e.g., Eckstein et al., InternationalPublication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565;Pieken et al., 1991 Science 253, 314; Usman and Cedergren, 1992 Trendsin Biochem. Sci. 17, 334; Usman et al., International Publication No. WO93/15187; and Rossi et al., Intemational Publication No. WO 91/03162, aswell as Usman, N. et al. U.S. patent application Ser. No. 07/829,729,and Sproat, European Patent Application 92110298.4 which describevarious chemical modifications that can be made to the sugar moieties ofenzymatic RNA molecules. Modifications which enhance their efficacy incells, and removal of stem II bases to shorten RNA synthesis times andreduce chemical requirements (All these publications are herebyincorporated by reference herein).

Administration of Ribozyme

Sullivan et al., PCT WO94/02595, describes the general methods fordelivery of enzymatic RNA molecules. Ribozymes may be administered tocells by a variety of methods known to those familiar to the art,including, but not restricted to, encapsulation in liposomes, byiontophoresis, or by incorporation into other vehicles, such ashydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesivemicrospheres. For some indications, ribozymes may be directly deliveredex vivo to cells or tissues with or without the aforementioned vehicles.Alternatively, the RNA/vehicle combination is locally delivered bydirect injection or by use of a catheter, infusion pump or stent. Otherroutes of delivery include, but are not limited to, intravascular,intramuscular, subcutaneous or joint injection, aerosol inhalation, oral(tablet or pill form), topical, systemic, ocular, intraperitoneal and/orintrathecal delivery. More detailed descriptions of ribozyme deliveryand administration are provided in Sullivan et al., supra and Draper etal, PCT WO93/23569 which have been incorporated by reference herein.

EXAMPLES

The following are non-limiting examples showing the synthesis ofnon-nucleotide mimetic-containing catalytic nucleic acids usingnon-nucleotide phosphoramidites.

As shown in FIG. 7, such non-nucleotides can be located in the bindingarms, core or the loop adjacent stem II of a hammerhead type ribozyme.Those in the art following the teachings herein can determine optimallocations in these regions. Surprisingly, abasic moieties can be locatedin the core of such a ribozyme.

Example 1 Synthesis of Hammerhead Ribozymes Containing Non-nucleotideMimetics: Polyether Spacers

Polyether spacers, compound 1 (FIG. 6; X=0, n=2 or 4), have beenincorporated both singly, n=2 or 4, or doubly, n=2, at the base of stemII of a hammerhead ribozyme, replacing loop 2, and shown to produce aribozyme which has lower catalytic efficiency. The method of synthesisused followed the procedure for normal RNA synthesis as described inUsman et al., J. Am. Chem. Soc. 1987, 109:7845 and in Scaringe et al.,Nucleic Acids Res. 1990, 18:5433, and makes use of common nucleic acidprotecting and coupling groups, such as dimethoxytrityl at the 5'-end,and phosphoramidites at the 3'-end. The average stepwise coupling yieldswere >98%. The design of these types of mimetics has not been optimizedto date, but, as discussed above, this can be readily achieved usingstandard experimental techniques. These experiments indicate thepotential of such mimetics to replace the loops and portions of stems inribozymes while maintaining catalytic activity. These mimetics may beincorporated not only into hammerhead ribozymes, but also into hairpin,hepatitis delta virus, or Group 1 or Group 2 introns. They are,therefore, of general use as replacement motifs in any nucleic acidstructure. Use of such mimetics allows about 2-10 nucleotides to beomitted from the final nucleic acid molecule compared to the use of anoligonucleotide without a non-nucleotide mimetic.

Example 2 Synthesis of Hammerhead Ribozymes Containing Non-nucleotideMimetics: Aromatic Spacers

In another example, a specific linker for the base of the stem II C-G ofa hammerhead ribozyme was designed. Applicant believes that the distancebetween the C1' carbons of the C-G base pair is about 16 Angstroms. Tojoin these two pieces of RNA by a covalent analog of the C-G base pair anew type of dimer phosphoramidite containing a linker between the 3'-OHand the 5'-OH of the G and C residues respectively can be constructed.Two types of base-pair mimetic are the rigid aromatic spacers, 2 or 3,shown in FIG. 6. These have been incorporated at the base of stem II ofa hammerhead ribozyme as described in Example 1, replacing loop 2, andshown to produce a ribozyme which has lower catalytic efficiency.Another mimetic is a flexible alkyl spacer similar to the polyamidebackbone described by Nielsen et al., Science 1991, 254:1497 (see, FIG.6; 6 or a derivative thereof 6a; Zuckerman et al., J. Am. Chem. Soc.1992, 114:10646). Use of such mimetics allows about 2-10 nucleotides tobe omitted from the final nucleic acid molecule compared to the use ofan oligonucleotide without a non-nucleotide mimetic.

Example 3 Synthesis of Non-nucleotide Mimetics Aromatic SpacerPhosphoramidite 2

This compound was originally described by Salunkhe et al., J. Am. Chem.Soc. 1992, 114:8768. The synthesis was modified as follows: Toterphthalic acid (1.0 g, 6.0 mmol) in DMF (12 mL) was added EDC (2.54 g,13.2 mmol), aminohexanol (1.55 g, 13.2 mmol) and N-methylmorpholine(1.45 mL, 13.2 mmol). The reaction mixture was stirred overnight atwhich time the solution was cloudy. Water was added to the reactionmixture to precipitate out the product. The solid was filtered andwashed with water and dried to provide 562 mg (25.7%) of the diol.

To the diol (250 mg, 0.687 mmol) in DMSO (40 mL) was added triethylamine(287 μL, 2.06 mmol), dimethoxytrityl chloride (220 mg, 0.653 mmol) andcatalytic DMAP. The reaction mixture was heated to 40° C. and stirredovernight. The mixture was then cooled to room temperature (about 20-25°C.), quenched with water and extracted three times with EtOAc. A solidprecipitate remained in the organic layer that was isolated and found tobe starting diol (50 mg, 20%). The organic layer was dried over Na₂ SO₄and evaporated. The resulting oil was purified with flash chromatography(10% EtOAc in hexanes to 100% EtOAc) to yield 250 mg (55%) of themonotritylated compound.

To the alcohol (193 mg, 0.29 mmol) in THF (1 mL) at 0° C. was addeddiisopropylethylamine (101 μL, 0.58 mmol) and then 2-cyanoethylN,N-diisopropylamino chlorophosphoramidite (78 μL, 0.35 mmol) dropwise.The resulting mixture was stirred for 5 minutes and then warmed to roomtemperature. After 1 hour the reaction mixture was quenched withmethanol and evaporated. The resulting oil was purified by flashchromatography (1:1 hexanes:EtOAc) to yield 158 mg (63%) of thephosphoramidite.

Example 4 Synthesis of Non-nucleotide Mimetics Aromatic SpacerPhosphoramidite 3

Referring to FIG. 8, to 3, 4, 9, 10-perylenetetracarboxylic dianhydride11 (1.0 g, 2.55 mmol) in quinoline (10 mL) was added ethanolamine (919μL, 15.3 mmol) and ZnOAc.2.5 H₂ O (140 mg, 0.638 mmol). The reactionmixture was heated to 190° C. for 8 hours. The solution was then cooled,1N HCl added to precipitate the product and the mixture was filtered.The solid was washed with hot 10% CaCO₃ until the filtrate was no longerpale green. The remaining bright red precipitate 12 was then dried.

The resulting diol 12 was then treated as outlined above for 2 toprovide the phosphoramidite 3.

Example 5 Synthesis of Hammerhead Ribozymes Containing Non-nucleotideMimetics: Abasic Nucleotides 4 and 5

Compound 4, R═H, was prepared according to lyer et al., Nucleic AcidsRes. 1990, 18:2855. Referring to FIG. 9A, compounds 4 and 5(R═O-t-butyidimethylsilyl) phosphoramidites were prepared as follows:

To a solution of D-ribose (20.0 g, 0.105 mol) in N,N-dimethylformamide(250 mL) was added 2,2-dimethoxypropane (50 mL) and p-toluenesulfonicacid monohydrate (300 mg). The reaction mixture was stirred for 16 hoursat room temperature and then evaporated to dryness. The crude productwas coevaporated with pyridine (2×150 mL), dissolved in dry pyridine(300 mL) and 4,4'-dimethoxytrityl chloride (37.2 g, 0.110 mol) was addedand stirred for 24 hours at room temperature. The reaction mixture wasdiluted with methanol (50 mL) and evaporated to dryness. The residue wasdissolved in chloroform (800 mL) and washed with 5% NaHCO₃ (2×200 mL),brine (300 mL), dried, evaporated, coevaporated with toluene (2×100 mL)and purified by flash chromatography in CHCl₃ to yield 40.7 g (78.1%) ofcompound a.

To a solution of dimethoxytrityl derivative a (9.0 g, 18.3 mmol) andDMAP (4.34 g, 36 mmol) in dry CH₃ CN, phenoxythiocarbonyl chloride (3.47g, 20.1 mmol) was added dropwise under argon. The reaction mixture wasleft for 16 hours at room temperature, then evaporated to dryness. Theresulting residue was dissolved in chloroform (200 mL), washed with 5%NaHCO₃, brine, dried, evaporated and purified by flash chromatography inCHCl₃, to yield 8.0 g (69.5%) of compound b as the β-anomer.

To a solution of intermediate b (3.0 g, 4.77 mmol) in toluene (50 mL)was added AIBN (0.82 g, 5.0 mmol) and Bu₃ SnH (1.74 g, 6.0 mmol) underargon and the reaction mixture was kept at 80° C. for 7 hours. Thesolution was evaporated and the resulting residue purified by flashchromatography in CHCl₃ to yield 1.5 g (66%) of protected ribitol C.

Subsequent removal of all protecting groups by acid treatment andtritylation provided the protected ribitol d which was then converted totarget phosphoramidites 4 and 5 by the general method described inScaringe et al., Nucleic Acids Res. 1990, 18:5433.

The synthesis of 1-deoxy-D-ribofuranose phosphoramidite 9 is shown inFIG. 9B. Our initial efforts concentrated on the deoxygenation ofsynthon 1, prepared by a "one pot" procedure from D-ribose.Phenoxythiocarbonylation of acetonide 1 under Robins conditions led tothe β-anomer 2 (J₁,2 =1.2 Hz) in modest yield (45-55%). Radicaldeoxygenation using Bu₃ SnH/AIBN resulted in the formation of theribitol derivative 3 in 50% yield. Subsequent deprotection with 90% CF₃COOH (10 m) and introduction of a dimethoxytrityl group led to the keyintermediate 4 in 40% yield (Yang et al., Biochemistry 1992, 31,5005-5009; Perreault et al., Biochemistry 1991, 30, 4020-4025; Paolellaet al., EMBO J. 1992, 11, 1913-1919; Peiken et al., Science 1991, 253,314-317).

The low overall yield of this (FIG. 9B) route prompted us to investigatea different approach to 4 (FIG. 9B). Phenylthioglycosides, successfullyemployed in the Keck reaction, appeared to be an alternative. However,it is known that free-radical reduction of the corresponding glycosylbromides with participating acyl groups at the C2-position can result inthe migration of the 2-acyl group to the C1-position (depending on Bu₃SnH concentration). Therefore we subjected phenylthioglycoside 5 toradical reduction with Bu₃ SnH (6.1 eq.) in the presence of Bz₂ O₂ (2eq.) resulting in the isolation of tribenzoate 6 in 63% yield (FIG. 9B).Subsequent debenzoylation and dimethoxytritylation led to synthon 4 in70% yield. Introduction of the TBDMS group, using standard conditions,resulted in the formation of a 4:1 ratio of 2- and 3-isomers 8 and 7.The two regioisomers were separated by silica gel chromatography. The2-O-t-butyldimethylsilyl derivative 8 was phosphitylated to providephosphoramidite 9 in 82% yield.

Example 6

Referring to FIGS. 10a and 10b the cleavage of substrate is shown byvarious modified ribozymes compared to unmodified ribozyme at 8 nM and40 nM concentrations. Specifically, a control ribozyme of sequenceucuccA UCU GAU GAG GCC GAA AGG CCG AAA Auc ccU (where lower caseincludes a 2' O-methyl group) was compared to ribozyme A (ucu ccA UCUGAU GAG GCC SGG CCG AAA Auc ccu), B (ucu ccA UCU GAU GAG CSG CG AAA Aucccu), C (ucu ccA UCU GAU GAG GCC bbb bGG CCG AAA Auc ccu), and D (ucuccA UCU GAU GAG Cbb bbG CGAA AAu ccc u) (where S=hexaethylene glycollinker); and b=abasic nucleotide 4). All were active in cleavingsubstrate.

Example 7 RNA Cleavage Assay in Vitro

Ribozymes and substrate RNAs were synthesized as described above.Substrate RNA was 5' end-labeled using [γ-³² P] ATP and T4polynucleotide kinase (US Biochemicals). Cleavage reactions were carriedout under ribozyme "excess" conditions. Trace amount (≦1 nM) of 5'end-labeled substrate and 40 nM unlabeled ribozyme were denatured andrenatured separately by heating to 90° C. for 2 min and snap-cooling onice for 10-15 min. The ribozyme and substrate were incubated,separately, at 37° C. for 10 min in a buffer containing 50 mM Tris-HCland 10 mM MgCl₂. The reaction was initiated by mixing the ribozyme andsubstrate solutions and incubating at 37° C. Aliquots of 5 μl are takenat regular intervals of time and the reaction quenched by mixing with anequal volume of 2X formamide stop mix. The samples were resolved on 20%denaturing polyacrylamide gels. The results were quantified andpercentage of target RNA cleaved is plotted as a function of time.

Referring to FIG. 11 there is shown the general structure of ahammerhead ribozyme targeted against site A (HHA) with various basesnumbered. Various substitutions were made at several of the nucleotidepositions in HHA. Specifically referring to FIG. 12, substitutions weremade at the U4 and U7 positions marked as X4 and X7 and also in loop IIin the positions marked by an X. The RNA cleavage activity of thesesubstituted ribozymes is shown in the following figures. Specifically,FIG. 13 shows cleavage by an abasic substituted U4 and an abasicsubstituted U7. As will be noted, abasic substitution at U4 or U7 doesnot significantly affect cleavage activity. In addition, inclusion ofall abasic moieties in stem II loop does not significantly reduceenzymatic activity as shown in FIG. 14. Further, inclusion of a 3'inverted deoxyribos does not inactivate the RNA cleavage activity asshown in FIG. 15.

Example 8 Smooth Muscle Cell Proliferation Assay

Hammerhead ribozyme (HHB) is targeted to a unique site (site B) withinc-myb mRNA. Expression of c-myb protein has been shown to be essentialfor the proliferation of rat smooth muscle cell (Brown et al., 1992 J.Biol. Chem. 267,4625).

The ribozymes that cleaved site B within c-myb RNA described above wereassayed for their effect on smooth muscle cell proliferation. Ratvascular smooth muscle cells were isolated and cultured as described(Stnchcomb et al., supra). These primary rat aortic smooth muscle cells(RASMC) were plated in a 24-well plate (5×103 cells/well) and incubatedat 37° C. in the presence of Dulbecco's Minimal Essential Media (DMEM)and 10% serum for ˜16 hours.

These cells were serum-starved for 48-72 hours in DMEM (containing 0.5%serum) at 37° C. Following serum-starvation, the cells were treated withlipofectamine (LFA)-complexed ribozymes (100 nM ribozyme was complexedwith LFA such that LFA:ribozyme charge ration is 4:1).

Ribozyme:LFA complex was incubated with serum-starved RASMC cells forfour hours at 37° C. Following the removal of ribozyme:LFA complex fromcells (after 4 hours), 10% serum was added to stimulate smooth cellproliferation. Bromo-deoxyuridine (BrdU) was added to stain the cells.The cells were stimulated with serum for 24 hours at 37° C.

Following serum-stimulation, RASMC cells were quenched with hydrogenperoxide (0.3% H₂ O₂ in methanol) for 30 min at 4° C. The cells werethen denatured with 0.5 ml 2N HCl for 20 min at room temperature. Horseserum (0.5 ml) was used to block the cells at 40° C. for 30 min up to˜16 hours.

The RASMC cells were stained first by treating the cells with anti-BrdU(primary) antibody at room temperature for 60 min. The cells were washedwith phosphate-buffered saline (PBS) and stained with biotinylatedaffinity-purified anti-mouse IgM (Pierce, USA) secondary antibody. Thecells were counterstained using avidin-biotinylated enzyme complex (ABC)kit (Pierce, USA).

The ratio of proliferating:non-proliferating cells was determined bycounting stained cells under a microscope. Proliferating RASMCs willincorporate BrdU and will stain brown. Non-proliferating cells do notincorporate BrdU and will stain purple.

Referring to FIG. 16 there is shown a ribozyme which cleaves the site Breferred to as HHB. Substitutions of abasic moieties in place of U4 asshown in FIG. 17 provided active ribozyme as shown in FIG. 18 using theabove-noted rat aortic smooth muscle cell proliferation assay.

Administration of Ribozyme

Selected ribozymes can be administered prophylactically, to viralinfected patients or to diseased patients, e.g., by exogenous deliveryof the ribozyme to a relevant tissue by means of an appropriate deliveryvehicle, e.g., a liposome, a controlled release vehicle, by use ofiontophoresis, electroporation or ion paired molecules, or covalentlyattached adducts, and other pharmacologically approved methods ofdelivery. Routes of administration include intramuscular, aerosol, oral(tablet or pill form), topical, systemic, ocular, intraperitoneal and/orintrathecal.

The specific delivery route of any selected ribozyme will depend on theuse of the ribozyme. Generally, a specific delivery program for eachribozyme will focus on unmodified ribozyme uptake with regard tointracellular localization, followed by demonstration of efficacy.Alternatively, delivery to these same cells in an organ or tissue of ananimal can be pursued. Uptake studies will include uptake assays toevaluate cellular ribozyme uptake, regardless of the delivery vehicle orstrategy. Such assays will also determine the intracellular localizationof the ribozyme following uptake, ultimately establishing therequirements for maintenance of steady-state concentrations within thecellular compartment containing the target sequence (nucleus and/orcytoplasm). Efficacy and cytotoxicity can then be tested. Toxicity willnot only include cell viability but also cell function.

Some methods of delivery that may be used include:

a. encapsulation in liposomes,

b. transduction by retroviral vectors,

c. conjugation with cholesterol,

d. localization to nuclear compartment utilizing antigen binding ornuclear targeting site found on most snRNAs or nuclear proteins,

e. neutralization of charge of ribozyme by using nucleotide derivatives,and

f. use of blood stem cells to distribute ribozymes throughout the body.

Delivery strategies useful in the present invention, include: ribozymemodifications, and particle carrier drug delivery vehicles. Unmodifiedribozymes, like most small molecules, are taken up by cells, albeitslowly. To enhance cellular uptake, the ribozyme may be modifiedessentially at random, in ways which reduce its charge but maintainsspecific functional groups. This results in a molecule which is able todiffuse across the cell membrane, thus removing the permeabilitybarrier.

Modification of ribozymes to reduce charge is just one approach toenhance the cellular uptake of these larger molecules. The randomapproach, however, is not advisable since ribozymes are structurally andfunctionally more complex than small drug molecules. The structuralrequirements necessary to maintain ribozyme catalytic activity are wellunderstood by those in the art. These requirements are taken intoconsideration when designing modifications to enhance cellular delivery.The modifications are also designed to reduce susceptibility to nucleasedegradation. Both of these characteristics should greatly improve theefficacy of the ribozyme. Cellular uptake can be increased by severalorders of magnitude without having to alter the phosphodiester linkagesnecessary for ribozyme cleavage activity.

Use

Those in the art will recognize that these ribozymes can be used inplace of other enzymatic RNA molecules for both in vitro and in vivouses well known in the art. See Draper WO 93/23569 and Sullivan WO94/12516.

Chemical modifications of the phosphate backbone will reduce thenegative charge allowing free diffusion across the membrane. Thisprinciple has been successfully demonstrated for antisense DNAtechnology. The similarities in chemical composition between DNA and RNAmake this a feasible approach. In the body, maintenance of an externalconcentration will be necessary to drive the diffusion of the modifiedribozyme into the cells of the tissue. Administration routes which allowthe diseased tissue to be exposed to a transient high concentration ofthe drug, which is slowly dissipated by systemic adsorption arepreferred. Intravenous administration with a drug carrier designed toincrease the circulation half-life of the ribozyme can be used. The sizeand composition of the drug carrier restricts rapid clearance from theblood stream. The carrier, made to accumulate at the site of infection,can protect the ribozyme from degradative processes.

Drug delivery vehicles are effective for both systemic and topicaladministration. They can be designed to serve as a slow releasereservoir, or to deliver their contents directly to the target cell. Anadvantage of using direct delivery drug vehicles is that multiplemolecules are delivered per uptake. Such vehicles have been shown toincrease the circulation half-life of drugs which would otherwise berapidly cleared from the blood stream. Some examples of such specializeddrug delivery vehicles which fall into this category are liposomes,hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesivemicrospheres.

From this category of delivery systems, liposomes are preferred.Liposomes increase intracellular stability, increase uptake efficiencyand improve biological activity.

Liposomes are hollow spherical vesicles composed of lipids arranged in asimilar fashion as those lipids which make up the cell membrane. Theyhave an internal aqueous space for entrapping water soluble compoundsand range in size from 0.05 to several microns in diameter. Severalstudies have shown that liposomes can deliver RNA to cells and that theRNA remains biologically active.

For example, a liposome delivery vehicle originally designed as aresearch tool, Lipofectin, has been shown to deliver intact mRNAmolecules to cells yielding production of the corresponding protein. Inanother study, an antibody targeted liposome delivery system containingan RNA molecule 3,500 nucleotides in length and antisense to astructural protein of HIV, inhibited virus proliferation in a sequencespecific manner. Not only did the antibody target the liposomes to theinfected cells, but it also triggered the internalization of theliposomes by the infected cells. Triggering the endocytosis is usefulfor viral inhibition. Finally, liposome delivered synthetic ribozymeshave been shown to concentrate in the nucleus of H9 (an example of anHIV-sensitive cell) cells and are functional as evidenced by theirintracellular cleavage of the sequence. Liposome delivery to other celltypes using smaller ribozymes (less than 142 nucleotides in length)exhibit different intracellular localizations.

Liposomes offer several advantages: They are non-toxic and biodegradablein composition; they display long circulation half-lives; andrecognition molecules can be readily attached to their surface fortargeting to tissues. Finally, cost effective manufacture ofliposome-based pharmaceuticals, either in a liquid suspension orlyophilized product, has demonstrated the viability of this technologyas an acceptable drug delivery system.

Other controlled release drug delivery systems, such as nonoparticlesand hydrogels may be potential delivery vehicles for a ribozyme. Thesecarriers have been developed for chemotherapeutic agents andprotein-based pharmaceuticals, and consequently, can be adapted forribozyme delivery.

Topical administration of ribozymes is advantageous since it allowslocalized concentration at the site of administration with minimalsystemic adsorption. This simplifies the delivery strategy of theribozyme to the disease site and reduces the extent of toxicologicalcharacterization. Furthermore, the amount of material to be applied isfar less than that required for other administration routes. Effectivedelivery requires the ribozyme to diffuse into the infected cells.Chemical modification of the ribozyme to neutralize negative charge maybe all that is required for penetration. However, in the event thatcharge neutralization is insufficient, the modified ribozyme can beco-formulated with permeability enhancers, such as Azone or oleic acid,in a liposome. The liposomes can either represent a slow releasepresentation vehicle in which the modified ribozyme and permeabilityenhancer transfer from the liposome into the infected cell, or theliposome phospholipids can participate directly with the modifiedribozyme and permeability enhancer in facilitating cellular delivery. Insome cases, both the ribozyme and permeability enhancer can beformulated into a suppository formulation for slow release.

Ribozymes may also be systematically administered. Systemic absorptionrefers to the accumulation of drugs in the blood stream followed bydistribution throughout the entire body. Administration routes whichlead to systemic absorption include: intravenous, subcutaneous,intraperitoneal, intranasal, intrathecal and ophthalmic. Each of theseadministration routes expose the ribozyme to an accessible diseasedtissue. Subcutaneous administration drains into a localized lymph nodewhich proceeds through the lymphatic network into the circulation. Therate of entry into the circulation has been shown to be a function ofmolecular weight or size. The use of a liposome or other drug carrierlocalizes the ribozyme at the lymph node. The ribozyme can be modifiedto diffuse into the cell, or the liposome can directly participate inthe delivery of either the unmodified or modified ribozyme to the cell.This method is particularly useful for treating AIDS using anti-HIVribozymes.

Also preferred in AIDS therapy is the use of a liposome formulationwhich can deliver oligonucleotides to lymphocytes and macrophages. Thisoligonucleotide delivery system inhibits HIV proliferation in infectedprimary immune cells. Whole blood studies show that the formulation istaken up by 90% of the lymphocytes after 8 hours at 37° C. Preliminarybiodistribution and pharmacokinetic studies yielded 70% of the injecteddose/gm of tissue in the spleen after one hour following intravenousadministration. This formulation offers an excellent delivery vehiclefor anti-AIDS ribozymes for two reasons. First, T-helper lymphocytes andmacrophages are the primary cells infected by the virus, and second, asubcutaneous administration delivers the ribozymes to the residentHIV-infected lymphocytes and macrophages in the lymph node. Theliposomes then exit the lymphatic system, enter the circulation, andaccumulate in the spleen, where the ribozyme is delivered to theresident lymphocytes and macrophages.

Intraperitoneal administration also leads to entry into the circulation,with once again, the molecular weight or size of the ribozyme-deliveryvehicle complex controlling the rate of entry.

Liposomes injected intravenously show accumulation in the liver, lungand spleen. The composition and size can be adjusted so that thisaccumulation represents 30% to 40% of the injected dose. The remainingdose circulates in the blood stream for up to 24 hours.

The chosen method of delivery should result in cytoplasmic accumulationin the afflicted cells and molecules should have somenuclease-resistance for optimal dosing. Nuclear delivery may be used butis less preferable. Most preferred delivery methods include liposomes(10-400 nm), hydrogels, controlled-release polymers, microinjection orelectroporation (for ex vivo treatments) and other pharmaceuticallyapplicable vehicles. The dosage will depend upon the disease indicationand the route of administration but should be between 100-200 mg/kg ofbody weight/day. The duration of treatment will extend through thecourse of the disease symptoms, usually at least 14-16 days and possiblycontinuously. Multiple daily doses are anticipated for topicalapplications, ocular applications and vaginal applications. The numberof doses will depend upon disease delivery vehicle and efficacy datafrom clinical trials.

Establishment of therapeutic levels of ribozyme within the cell isdependent upon the rate of uptake and degradation. Decreasing the degreeof degradation will prolong the intracellular half-life of the ribozyme.Thus, chemically modified ribozymes, e.g., with modification of thephosphate backbone, or capping of the 5' and 3' ends of the ribozymewith nucleotide analogues may require different dosaging. Descriptionsof useful systems are provided in the art cited above, all of which ishereby incorporated by reference herein.

For a more detailed description of ribozyme design, see, Draper, U.S.Ser. No. 08/103,243 filed Aug. 6, 1993, hereby incorporated by referenceherein in its entirety.

Other embodiments are within the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:       - # 21                                - -  - - (2) INFORMATION FOR SEQ ID NO:  1:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         36 base pairs                                     (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 1:                          - - UCUCCAUCUG AUGAGGCCGA AAGGCCGAAA AUCCCU      - #                       - #       36                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:  2:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         33 base pairs                                     (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for                             hexaethylene - # glycol linker.                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 2:                          - - UCUCCAUCUG AUGAGGCCNG GCCGAAAAUC CCU       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:  3:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         29 base pairs                                     (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for                              hexaethylene - # glycol linker.                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 3:                          - - UCUCCAUCUG AUGAGCNGCG AAAAUCCCU         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:  4:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         36 base pairs                                     (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for                              abasic nu - #cleotide 4.                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 4:                          - - UCUCCAUCUG AUGAGGCCNN NNGGCCGAAA AUCCCU      - #                  -     #       36                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:  5:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         32 base pairs                                     (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for                              abasic nu - #cleotide 4.                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 5:                          - - UCUCCAUCUG AUGAGCNNNN GCGAAAAUCC CU       - #                  - #              32                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:  6:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         11 base pairs                                     (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for any          base.                                                                                          The lette - #r "H" stands for C, A or U.                        - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  6:                        - - NNNNUHNNNN N               - #                  - #                      - #       11                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:  7:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         28 base pairs                                     (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for any          base.                                                                            - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  7:                                      NNNNNCUGAN - #GAGNNNNNNC GAAANNNN             - #                      - #     28                                                            - - (2) INFORMATION FOR SEQ ID NO:  8:                                        - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         15 base pairs                                     (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for any          base.                                                                                          The lette - #r "Y" stands for U or C.                                         The lette - #r "H" stands for A, U or C.                        - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  8:                        - - NNNNNNNYNG HYNNN              - #                  - #                      - #    15                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:  9:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         47 base pairs                                     (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for any          base.                                                                            - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  9:                        - - NNNNGAAGNN NNNNNNNNNA AAHANNNNNN NACAUUACNN NNNNNNN   - #                    47                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:  10:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         85 base pairs                                     (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  10:                       - - UGGCCGGCAU GGUCCCAGCC UCCUCGCUGG CGCCGGCUGG GCAACAUUCC GA -             #GGGGACCG     60                                                                 - - UCCCCUCGGU AAUGGCGAAU GGGAC          - #                  - #                   85                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:  11:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         176 base pairs                                    (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  11:                       - - GGGAAAGCUU GCGAAGGGCG UCGUCGCCCC GAGCGGUAGU AAGCAGGGAA CU -             #CACCUCCA     60                                                                 - - AUUUCAGUAC UGAAAUUGUC GUAGCAGUUG ACUACUGUUA UGUGAUUGGU AG -            #AGGCUAAG    120                                                                 - - UGACGGUAUU GGCGUAAGUC AGUAUUGCAG CACAGCACAA GCCCGCUUGC GA - #GAAU            176                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  12:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         13 base pairs                                     (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 12:                         - - GACCGUCAGA CGC              - #                  - #                      - #      13                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:  13:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         32 base pairs                                     (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 13:                         - - GCUGGUCUGA UGAGGUCCGG ACCGAAACGG UC       - #                  - #              32                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:  14:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         15 base pairs                                     (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 14:                         - - AGGGAUUAAU GGAGA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:  15:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         32 base pairs                                     (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 15:                         - - UCUCCAUCUG AUGAGGGAAA CCGAAAAUCC CU       - #                  - #              32                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:  16:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         15 base pairs                                     (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 16:                         - - AGGGAUUAAU GGAGA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:  17:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         33 base pairs                                     (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for                              abasic su - #bstitutions.                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 17:                         - - UCUCCAUCNG ANGAGGNNNN CCGAAAAUCC CUN       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:  18:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         15 base pairs                                     (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 18:                         - - GGAGAAUUGG AAAAC              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:  19:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         34 base pairs                                     (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 19:                         - - GUUUUCCCUG AUGAGGGGAA ACCCGAAAUU CUCC       - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:  20:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         15 base pairs                                     (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 20:                         - - GGAGAAUUGG AAAAC              - #                  - #                      - #    15                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:  21:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         34 base pairs                                     (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #  The letter "N" stands for                              abasic su - #bstitutions.                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 21:                         - - GUUUUCCCNG AUGAGGGGAA ACCCGAAAUU CUCC       - #                  -      #        34                                                                   __________________________________________________________________________

What is claimed is:
 1. A nucleic acid molecule comprising an abasicmoiety at the 3' end or at the 5' end or at both the 3' end and the 5'end of said nucleic acid molecule, wherein said abasic moiety lacks anucleic acid base.
 2. The nucleic acid molecule of claim 1, wherein saidabasic moiety is an inverted abasic moiety.
 3. The nucleic acid moleculeof any of claims 1-2, wherein said abasic moiety comprises a sugarmodification.
 4. The nucleic acid molecule of claim 3, wherein saidsugar modification is 2'-H.
 5. The nucleic acid molecule of claim 3,wherein said sugar modification is 2'-O-methyl.
 6. The nucleic acidmolecule of any of claims 1-2, wherein said nucleic acid molecule is anenzymatic nucleic acid molecule.
 7. The nucleic acid molecule of any ofclaims 1-2, wherein said nucleic acid molecule is an antisense nucleicacid molecule.
 8. The nucleic acid molecule of any of claims 1-2,wherein said abasic moiety is present at the 3' end of the nucleic acidmolecule.
 9. The nucleic acid molecule of any of claims 1-2, whereinsaid abasic moiety is present at the 5' end of the nucleic acidmolecule.
 10. The nucleic acid molecule of any of claims 1-2, whereinsaid abasic nucleic acid moiety is present at the 3' end and the 5' endof said nucleic acid molecule.
 11. A cell including the nucleic acidmolecule of any of claims 1-2.
 12. The cell of claim 11, wherein saidcell is a mammalian cell.
 13. The cell of claim 12, wherein saidmammalian cell is a human cell.